We propose to investigate why central visual pathways fail to regenerate after injury and how their repair can be enhanced. Specifically, we will investigate why the axons of mature retinal ganglion cells (RGCs) fail to regenerate after axotomy. The regeneration of RGCs through the optic nerve has long served as a simple model system for study of CNS regenerative failure. The rat optic nerve consists primarily of RGC axons, astrocytes, and myelinating oligodendrocytes. We have developed methods to purity and culture rodent RGCs, optic nerve astrocytes and oligodendrocytes. Using these methods we will continue to investigate the molecular mechanisms that promote and inhibit RGC axon elongation in culture, and how we can apply this knowledge to enhance regeneration. In this proposal, we will investigate the molecular basis of 3 phenomena implicated in the failure of RGC axons to regenerate. Over the last grant period, we discovered that neonatal RGCs are signaled by amacrine cells to irreversibly lose their intrinsic competence to rapidly regenerate their axons. In the first aim, we will use gene profiling to investigate the molecular basis for this loss. In the second aim, we investigate the identity of myelin-associated inhibitors of regeneration in the optic nerve and the RGC receptors that they bind to by constructing bacteriophage display libraries of single chain antibodies and selecting antibodies that enhance RGC regeneration. In the third am, we will investigate why the clearance of myelin debris in Wallerian degeneration is so prolonged after optic nerve injury. We will specifically test the hypothesis that this is accounted for by the recent discovery that CNS microglia are immature myeloid precursor cells rather than quiescent phagocytes. Finally, we will apply what we learn in the first 3 aims to determine if we can enhance RGC regeneration after optic nerve injury in vivo. Our ultimate goal is to understand why RGCs fail to regenerate afar axotomy and to develop new treatments promote their regeneration after injury in ocular diseases including glaucoma, retinal ischemia, optic neuritis, and neuropathy.